PROJECT SUMMARY: PROJECT 3 The aberrant SS18-SSX fusion protein causes synovial sarcoma in humans and in mice. Normally these two proteins, SS18 and SSX, have distinct roles in regulating gene expression, but their fusion together leads to misregulation. SS18 functions as a member of a large chromatin remodeling complex termed the BAF complex. The BAF complex is an essential and abundant complex that normally functions in all cells as a chromatin remodeling machine; it removes repressive chromatin (i.e. nucleosomes and their modifiers) from genes, enabling their activation. However, the SS18-SSX fusion protein apparently alters the functions of the BAF complex, though precisely how BAF function is altered by the SS18-SSX fusion is not known. Among the possibilities are changes in BAF composition, targeting, or activity. This grant focuses on exploring the impact of the SS18-SSX fusion on BAF composition and activity. The long term objectives of Project 3 are to obtain a deep mechanistic understanding of how the SS18-SSX fusion protein alters BAF complex composition and activity and to exploit that information to develop new therapeutics, through the following three Aims: Aim 1. Determine the impact of BAF subunit participation, including SS18-SSX, on complex stability and nucleosome sliding and ejection. Approach. We will create and purify recombinant BAF complexes, with defined inclusion or exclusion of individual subunits?including SS18 or SS18-SSX?and will test the impact of each on in vitro nucleosome remodeling by the BAF complex, as well as validate the result in vivo. Aim 2. Characterize membership and subunit dependencies of BAF complexes in synovial sarcoma. Approach. Reverse genetic screens in cell lines will test the critical role of each BAF subunit to assemble with SS18-SSX and other BAF subunits, as well as assess tumor cell viability. Aim 3. Characterize and advance BRD9 as a therapeutic target in synovial sarcoma. Approach. BRD9 is a component specific to SS18-SSX-including BAFs in synovial sarcoma cells, which are sensitive to its degradation. Experiments will map the critical domains of BRD9, its specific interactome in synovial sarcoma cells, and the impact of its degradation or inhibition in vitro and in vivo. Together, these Aims will deliver major new information on how the SS18-SSX fusion protein misregulates the BAF complex, revealing new therapeutic strategies. It will also explore the promising BRD9 target through a set of approaches, advancing the work to the pre-clinical realm.